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PG production has long been a target for antibiotics, and its breakdown is a source for human immune recognition. A key component of bacterial PG, <jats:italic>N<\/jats:italic>\u2010acetyl muramic acid (NAM), is a vital element in many synthetically derived immunostimulatory compounds. However, the exact molecular details of these structures and how they are generated remain unknown due to a lack of chemical probes surrounding the NAM core. A robust synthetic strategy to generate bioorthogonally tagged NAM carbohydrate units is implemented. These molecules serve as precursors for PG biosynthesis and recycling. <jats:italic>Escherichia coli<\/jats:italic> cells are metabolically engineered to incorporate the bioorthogonal NAM probes into their PG network. The probes are subsequently modified using copper\u2010catalyzed azide\u2010alkyne cycloaddition to install fluorophores directly into the bacterial PG, as confirmed by super\u2010resolution microscopy and high\u2010resolution mass spectrometry. Here, synthetic notes for key elements of this process to generate the sugar probes as well as streamlined user\u2010friendly metabolic labeling strategies for both microbiology and immunological applications are described. \u00a9 2019 by John Wiley &amp; Sons, Inc.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Synthesis of peracetylated 2\u2010azido glucosamine<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Synthesis of 2\u2010azido and 2\u2010alkyne NAM<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Synthesis of 3\u2010azido NAM methyl ester<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Incorporation of NAM probes into bacterial peptidoglycan<\/jats:p><jats:p><jats:bold>Basic\u00a0Protocol 5<\/jats:bold>: Confirmation of bacterial cell wall remodeling by mass spectrometry<\/jats:p>","DOI":"10.1002\/cpch.74","type":"journal-article","created":{"date-parts":[[2019,10,10]],"date-time":"2019-10-10T13:51:46Z","timestamp":1570715506000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Metabolic Incorporation of <i>N<\/i>\u2010Acetyl Muramic Acid Probes into Bacterial Peptidoglycan"],"prefix":"10.1002","volume":"11","author":[{"given":"Kristen E.","family":"DeMeester","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry University of Delaware  Newark Delaware"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hai","family":"Liang","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry University of Delaware  Newark Delaware"},{"name":"\u00a0Cutaneous Microbiome and Inflammation Section, Dermatology Branch National Institute of Arthritis and Musculoskeletal and Skin Diseases  Bethesda 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